Ribonuclease digestion

As an example of the use of paper electrophoresis for the fractionation of oligonucleotides consider the fractionation of some decanucleotides obtained from MS2 viral RNA by T1 ribonuclease digestion. The specificity of the enzyme ensured that each oligonucleotide contained only one Gp residue and the decanucleotides were isolated by column chromatography ( 5.2.1.2). The oligonucleotide mixture was electrophoresed for 17 hr at 6 volt/cm in 0.02 M ammonia formate pH 2.7. At this pH the nucleotides, Ap, Cp, Gp, Up have net charges, q, of —0.08, —0.02, —0.66, 0.98 respectively. Thus the mobility of these decanucleotides should depend mostly on the ratio of Ap+Cp to Up in their composition. Good separation in the predicted order has been obtained (Rushizky et al. 1965). 

A similar system has been used by Rushizky and Sober (1962a) to fractionate the products of T1 ribonuclease digestion of RNA. The electrophoresis buffer was dilute formic acid pH 2.35 and the... 

Armstrong et at. (1964) used l-propanol water ammonia (55 35 10, v/v/v) in combination with paper electrophoresis to fractionate the products of pancreatic ribonuclease digestion of some specific transfer RNAs. 

The oligonucleotides in a pancreatic ribonuclease digest of RNA have the general formula (purine nucleotide) i pyrimidine nucleotide, n = 1, 2,3,... Thus for each value of the chain length, n nucleotides, the charge at neutral pH and the purine pyrimidine ratio are functions of n only and both increase monotonically with n. This... 

Fig. 5.1. Fractionation of heptanucleotides. Heptanucleotides were isolated from a pancreatic ribonuclease digest of l2 phage RNA by chromatography on DEAE-ceilulose. Fig. 4.1. They were dialysed against distilled water, loaded onto a 9 x 500 mm column of DEAE-Sephadex previously equilibrated with 7 M urea+ I mM EDTA + HCl to pH 2.7 and eluted with a linear gradient from 0 to 0.2 M NaCI in 7 M urea+1 mM EDTA + HCI to pH 2.7. The eluent was collected directly into polyethylene vials and the Cerenkov radiation from the label in each fraction was measured in the liquid scintillation counter.

These two columns, DEAE-cellulose at neutral pH followed by DEAE-Sephadex at pH 2.7, suffice to separate all the components with different nucleotide compositions present in complete pancreatic ribonuclease digests of RNA molecules with at least about 10 nucleotides before digestion. Sometimes, these fractions contain only... 

The DEAE-Sephadex pH 2.7 fractionation gives reproducible elution positions for oligonucleotides, and the nucleotide compositions of an oligonucleotide from a pancreatic ribonuclease digest can sometimes be deduced from its relative position on the chromatogram. 

More work is needed on the fractionation of T1 ribonuclease digests. The procedure of Rushizky et al. (1965) is to fractionate the mixture according to chain length on DEAE-Sephadex at pH 7.6... 

Fig. 10.11. Autoradiograph of partial Tl-ribonuclease digest of P-labeled R17 virus RNA. The bands indicated were cut out and used for sequence determination...

H.G. Zachau, G. Acs, F. Lipmann, Isolation of adenosine amino acid esters from a ribonuclease digest of soluble, liver ribonucleic acid. Proc. Natl. Acad. Sci. USA 44 885-889 (1958)... 

AmpliScribe T7 Flash Enzyme solution Ribonuclease Digestion Transcript 3-HPA... 

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